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Glycosylceramide analoguesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycosideGlycosylceramide analogues description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060116331, Glycosylceramide analogues. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit of Gandhi et al., U.S. Prov. Appl. No. 60/413,882, filed Sep. 27, 2002, and hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to novel glycolipids which have biological activity, e.g., the ability to modulate the immune system. More specifically, synthetic analogues of .alpha.-galactosylceramides are disclosed. These molecules have the potential to activate the immune cells by inducing the secretion of cytokines and modulate immune responses. The invention also relates to the therapeutic application of these molecules in immunotherapy, in particular as immunostimulatory adjuvants for vaccine development and as immunoinhibitory agents for the treatment of autoimmune diseases and inflammation. [0004] 2. Description of the Background Art [0005] As its name suggests, a glycosylceramide combines a carbohydrate moiety and a ceramide moiety. A ceramide, in turn, comprises the divalent residue of a sphingoid base (a long-chain aliphatic amino alcohol), and a monovalent fatty acyl moiety. More particularly, it is the result of acylating the amino nitrogen of the divalent residue (--O--CH2-CH(--NH--)--R') of a sphingoid base to obtain --O--CH2-CH(--NH-R'')--R' (where R' is alkyl or alkenyl, and may be hydroxylated, and where R'' is a fatty acyl group, --C(.dbd.O)--R.sup.a, where R.sup.a is substituted or unsubstituted alkyl). The galactosylceramide is thus the result of O-linking the Galactose to the residue of the ceramide, i.e., [0006] Galactose-O--CH2-CH(--NH-R'')-R' [0007] Galactosylceramides are the principal glycosphingolipids in brain tissue, and hence are also known as cerebrosides. Glucosylceramides are the principal glycosphingolipids in the photosynthetic tissues of plants. They are also found in animal tissues, for example, in skin lipids. Other glycosylceramides are known in nature. [0008] The naturally occurring sphingoid bases vary in terms of the length of the main carbon chain (usually 14-22 carbons), the number of double bonds (usually 0, 1, or 2; the double bonds may be cis or trans, and the location(s) can vary, e.g., C-4 in sphingosine and C-8 in dehydrophytosphingosine), and the number of hydroxyl groups (usually 2 or 3; note that in a galactosylceramide, one of these hydroxyl groups becomes --OR, where R is the Gal). They can have branched chains, e.g., with methyl substituents. Much if not all of this variation is also seen among the naturally occurring glycosylceramides. [0009] Among the naturally occurring ceramides, there is also variation in the length of the fatty acid moiety (usually 16-26, with some preference for even numbers), and in whether or not the fatty acid moiety is hydroxylated. [0010] Agelasphins, a family of .alpha.-galactosylceramides (.alpha.-GalCer, FIG. 1), were originally extracted from marine sponges and found to exhibit potent anti-tumor properties and other therapeutic applications (Natori et al. 1994). One of .alpha.-GalCer's synthetic analogues, KRN7000 (FIG. 1; compound 7 in FIG. 11) is a promising immunomodulatory agent, which is currently being evaluated for its potential benefits in antitumor and antiinfectious therapies as well as in the prevention of type I diabetes and autoimmune encephalomyelitis. The adjuvant effect of .alpha.-GalCer has also been demonstrated with various different immunogens by its ability to strongly enhance antigen-specific CD8.sup.+ T cell response (Gonzalez-Aseguinolaza et al. 2002). [0011] Peptide/glycopeptide antigens are processed and presented by antigen presenting cells (APC) in the context of MHC I or II to T cell receptors (TCRs). On the other hand, glycolipid antigens are bound to CD1 molecules and presented to TCR. CD1 molecules represent a new class of highly conserved, antigen presenting cell surface proteins (Park, S.-H. & Bendelac, A. Nature, 2000, 406, 788-792). They recognize and bind glycolipid antigens through lipid -protein interactions and present the sugar moiety of the antigen to a receptor on natural killer T-cells (NKT cells) to activate the immune system. In humans, five different isoforms of CD1 have been detected so far. In the case of .alpha.-GalCer, it binds to CD1d molecule and the complex is recognized at picomolar concentrations by the conserved semi-invariant, CD1d-restricted .alpha.b TCR of mouse and human NKT cells (Kawano et al. 1997). The nature and orientation of the polar head group of .alpha.-GalCer molecule are likely to be important for TCR contact, while the nature of the lipophilic group in the ceramide moiety modulates the binding of .alpha.-GalCer to CD1d molecule. [0012] .alpha.-GalCer and its analogues are known to induce cell proliferation and cytokine production by natural killer (NK) T cells. Recently it was demonstrated that activation of NK T cells by .alpha.-GalCer causes bystander activation of NK, B, CD4.sup.+, and CD8.sup.+ T cells (Gonzalez-Aseguinolaza et al. 2002). A unique property of .alpha.-GalCer is its ability to induce both Th1 and Th2 immunity, which in turn is effected by cytokines, e.g., interleukin-4 (IL-4) and interferon-gamma (IFN-.gamma.). Some .alpha.-GalCer analogues elicit substantial amount of both IL-4 and IFN-g, while others elicit one predominantly over the other. It is well understood in immunology that IL-4 supports humoral immune (Th2) responses, while IFN-.gamma. supports cellular immune (Th1) response. Compounds that elicit predominantly or exclusively IL-4 might be useful as therapeutic agents for Th1-mediated autoimmune diseases, such as inflammation, type I diabetic, and multiple sclerosis. On the other hand, compounds that predominantly elicit IFN-.gamma. might be useful in effective vaccine development against intra-cellular pathogens, such as malaria, tuberculosis, and cancers. [0013] .alpha.-GalCer is a glycolipid comprising a hydrophilic carbohydrate moiety with .alpha.-linkage to the hydrophobic ceramide portion consisting of a long fatty acyl chain (C.sub.26) N-linked to sphingosine base (C.sub.18). Molecular interaction of .alpha.-GalCer with CD1d is necessary for V.alpha.14 NKT cell activation. It is speculated that the ceramide portion binds to the floor of the hydrophobic cleft of CD1d, while the hydrophilic sugar moiety is likely to interact with the V.alpha.14/Vb8.2 receptor and/or .alpha.-helix of CD1d. Structure-activity relationship studies (Uchimura, A. et al. Bioorg. Med. Chem. 1997, 5, 1447; Uchimura, A. et al. Bioorg. Med. Chem. 1997, 5, 2245-2249; Costantino, V. et al. Tetrahedron, 1996, 52, 1573-1578; Morita, M. et al. J. Med. Chem. 1995, 38, 2176-2187; Kawano et al., Science, 1997, 278, 1616-1629) have shown that, [0014] the length of the carbon chains on the ceramide is important, because a shorter length of either the fatty acyl chains or the sphingosine base reduced its ability to cause V.alpha.14 NKT cell proliferation; [0015] the .alpha.-anomeric configuration of the inner sugar is very important for stimulation of V.alpha.14 NKT cells, as indicated by the fact that .beta.-GalCer does not stimulate V.alpha.14 NKT cells readily; in addition, many kinds of monoglycosylated .beta.-D-pyranosylceramides (lactosylceramide, etc.) occur naturally, but there is no report that these monoglycosylated .beta.-D-pyranosylceramides have marked immunostimulatory effects; [0016] the configuration of the 2-OH group of the sugar moiety is very important for stimulation of V.alpha.14 NKT cells because .alpha.-mannosylceramide (.alpha.-ManCer), having a different configuration of the 2-OH group of the sugar moiety from .alpha.-CalCer, failed to stimulate V.alpha.14 NKT cells; [0017] the configuration of the 4-OH of the sugar moiety is not important for the manifestation of NKT immunostimulatory activity, since .alpha.-glucosylceramide (.alpha.-GlcCer) readily stimulate V.alpha.4 NKT cells; [0018] the configuration of 6-OH group of the sugar moiety is less important for the manifestation of the NKT immunostimulatory activity; and [0019] the 3'-OH on the sphingosine is very important for NKT immunostimulatory activity, because .alpha.-GalCer lacking 3'-OH sphingosine has no effect. [0020] Collectively, both carbohydrate and ceramide moieties play important roles in the exhibition of biological activities of .alpha.-GalCer molecules. Since the recognition event is highly specific for glycolipids and no carrier proteins are required, this novel defense mechanism has gained considerable interest in the past years, with the hope that a new type of therapeutic agents, including vaccines, may be developed in the future. With our growing knowledge of how .alpha.-GalCers stimulate immune cells, our current interest focuses on the discovery of novel synthetic analogues of .alpha.-GalCer with biological activities similar to their natural counterparts. One specific interest is to design novel structures which can elicit predominantly Th2 cytokine(s), e.g. (IL-4), over Th2 cytokine(s), e.g. IFN-.gamma., or vise versa, so that selective therapeutic benefits can be found with these compounds based on their ability of inducing different cytokine profiles. [0021] Glycosylceramides with unsaturated fatty acyl moieties. Costantino, et al., Bioorgan. Med. Chem. Lett. 9: 271-6 (1999) discloses two glycosyl ceramides (compounds 2a and 2b, named plakoside A and B) in which the fatty acyl moiety (corresponding to R3 in our formula F-A) comprises a single alkenic double bond. Plakoside A and B were isolated from the Caribbean sponge Plakortis simplex. These "simplexides" are immunoinhibitory agents. [0022] Glycosylceramides are also known which have unsaturated sphingoid base moieties. The website www.lipid.co.uk/infores/Lipids/cmh refers to the existence of cerebrosides of seeds from scarlet runner beans and kidney beans whose sphingoid bases have the structures d18:2-4t,8t or d18:2-4t,8c. [0023] Glycosylceramide analogues with steroidal, terpenoidal or alkaloidal moieties. We are not aware of any naturally occurring or synthetic glycosylceramide analogues with steroidal, terpenoidal or alkaloidal moieties. In this regard, it should be noted that while AGL-597 contains biotin (AGL597, the biotinylated analogue of KRN7000, was reported by Sakai, et al., Organic Lett. 1: 359-61 (1999) ), and biotin contains heterocyclic nitrogen, we do not believe that the art normally identifies biotin as an alkaloid. However, to avoid any possibility of confusion, we have defined "alkaloid" to formally exclude biotin. [0024] Fluorinated glycosylceramide analogues. Fluorine occurs extremely rarely in biomolecules, mostly as a monofluorinated fatty acid, at the omega carbon. [0025] Fluorocarbons share many of the properties of the cognate hydrocarbons. For example, fluorinated analogs of natural compounds can still be recognized by the normal enzymes or receptors. Thus, fluorinated methylmethionine, tryptophan, phenylalanine and tyrosine are still recognized by cognate amino acyl-tRNA synthetases. See Marsh, E. Neil G., "Toward the nonstick egg: designing fluorous proteins", Chemistry & Biology 7:R153-R157 (2000). Indeed, fluorination can increase binding; trifluoroleucine syubstitution in melittin had enhanced affinity for lipid bilayer membranes. Niemz and Tirrell, "Self-association and membrane-binding behavior of melittins containing trifluoroleucine", J. Am. Chem. Soc. 123: 7407-13 (2001). [0026] The fluorocarbons are, however, much more hydrophobic than their cognate hydrocarbons. For example, trifluoromethyl is over twice as hydrophobic as methyl. Fluorination has been used to increase the lipophilicity, and hence bioavailability of drugs, as in the case of fenfluramine. However, while some fluorocarbons are hydrophobic, perfluorocarbons are poorly soluble in hydrocarbon solvents, leading one commenter to refer to them as being fluorophilic, rather than lipophilic. The synthesis of fluorous proteins has been suggested. See Marsh (2000). [0027] Faroux-Corlay, et al., "Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity", Carbohydr. Res., 327: 223-260 (2000), describes the synthesis of three series of fluorinated analogues of beta GalCer, and evaluation of their anti-HIV activity. Beta GalCer is an alternative receptor allowing HIV-1 entry into CD4(-)/GalCer(+) cells by recognition of the V3 loop of HIV gp120. [0028] In the first series, in the terms of our general formula A, R is beta-Gal, L is the native --CH2-CH<, R2 is H, and A' and R3 are as follows: TABLE-US-00001 A' R3 (their R2) --C(.dbd.O)--NH--(CH2)13CH3 --C(.dbd.O) (CH2)10C4F9 --C(.dbd.O)--NH--(CH2)15CH3 --C(.dbd.O) (CH2)10C6F13 --C(.dbd.O)--NH--(CH2)11C4F9 --C(.dbd.O) (CH2)10C6F13 [0029] In the second series, the group corresponding to R3 in our general formula F-A' is --C(.dbd.O) (CH2)4C6F13, while R2 is --(CH2)24-N(--C(.dbd.O)R3)-CH2CH2OH or --(CH2)24-N(--C(.dbd.O)R3)-CH2CH2O-- betaGal, R is betaGal, L is --CH2-CH<, and A' is --H. (Note that we do not allow all of these choices.) Continue reading about Glycosylceramide analogues... 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